Integrase (the 3rd retroviral enzyme) is a rationale drug target because it is required for viral replication and does not have a known cellular equivalent. Validation of integrase as a pharmacological target for AIDS treatment has recently been demonstrated by the successful clinical trials with two inhibitors, raltegravir (Merck) and elvitegravir (Gilead). Those two inhibitors have a common mechanism of action by blocking the strand transfer step of the integration reaction. They also share a diketo acid (DKA) motif, and are commonly referred to as strand transfer inhibitors (STI). Raltegravir is expected to be approved by the FDA later this year for relapsing and resistant AIDS patients. Integrase is encoded by the HIV provirus and can be expressed as an active recombinant protein. Our laboratory has pioneered the integrase inhibitors research field, discovered several families of lead inhibitors and patented some with the aim of therapeutic development. We are investigating the molecular mechanism of action of diketo acid (DKA) derivatives in collaboration with Dr. Terrence Burke (Laboratory of Medicinal Chemistry, CCR, NCI) and with Dr. Roberto DiSanto (University of Rome, Italy). We are also investigating their binding site in the integrase-DNA complex. We found that azido derivatives of diketo acids and bifunctional DKA are potent and selective anti-integrase inhibitors and are antiviral. Patent applications have been filed for our derivatives. Our current hypothesis is that DKA inhibitors bind at the interface of the integrase-viral DNA complex by forming a coordination complex with the divalent metal at the enzyme active catalytic site following the 3-processing step of the integration reaction. We have now extended this concept of chelation to a two new families of drugs, conocurvones (isolated from an Australian plant) and tropolones (isolated from Canadian trees). In addition, we recently found in collaboration with Dr. Peter Roller (Laboratory of Medicinal Chemistry, CCR, NCI) that cationic peptides derived from indolicidin are integrase inhibitors. Their mechanism of action is novel as they bind to DNA, and thereby interfere with the formation of competent integrase-DNA complexes. In parallel, we have developed new integrase-DNA crosslinking assays using modified DNA substrates and mutant enzymes to determine the drug binding sites. We have implemented a novel high-throughput assay based on electrochemiluminescence (BioVeris). Using recombinant HIV1 integrase, we are screening chemical libraries to keep discovering novel inhibitors with original structures that will overcome resistance to DKA. The compounds are prioritized in collaboration with our colleagues from the NCI-CCR Laboratory of Medicinal Chemistry, the HIV Drug Resistance Program and the HIV and AIDS Malignancy Branch. Highest priority compounds are examined for mechanism of action, antiviral activity and structure-activity relationship with the aim of optimizing the drugs for medicinal development. We have filed patent application for tropolones and diketo acid derivatives as anti-integrase inhibitors and anti-HIV drugs. Additional compounds with structures differing from know inhibitors are being pursued with the aim of providing novel anti-integrase drugs that can be patented and developed as new therapeutics against HIV and AIDS. The novel compounds will also be added to our pharmacophore collection and used to select compounds to be screened from other chemical libraries (ChemNavigator and NIH Roadmap initiative).